ABSTRACT Voltage-gated sodium (Nav) channels initiate action potentials in the heart, and voltage-gated calcium (Cav) channels initiate excitation-contraction coupling. They are related proteins with a common evolutionary ancestor, and they are molecular targets for Class I and Class IV antiarrhythmic drugs (AADs) used in control of life-threatening cardiac arrhythmias. The structural basis for AAD action is unknown. We have determined the crystal structure of an ancestral bacterial Nav channel (NavAb) at 2.7 resolution and revealed the structural basis for voltage sensing, pore opening and closing, ion selectivity, and slow inactivation. This structure also revealed fenestrations that lead laterally from the lipid bilayer into the pore and provide an access pathway for entry of pore-blocking AADs. We constructed a Ca-selective form of NavAb, termed CavAb, and used this construct to reveal the structural basis for Ca selectivity at atomic resolution. We are now focusing on the structural basis for state-dependent block of Nav and Cav channels by AADs. CavAb is blocked by all three structural subclasses of Class IV AADs in a state-dependent manner with nM affinity. We found that the phenylalkylamine verapamil binds to a receptor site in the pore, at the inner end of the ion selectivity filter, and physically blocks it. In contrast, amlodipine and other dihydropyridines bind at a site on the lipid-facing outer surface of the pore module, at the interface between two voltage-sensing modules, and allosterically block the pore. These results reveal drug-receptor complexes of Cav channels for the first time and set the stage for complete analysis of the mechanism of state-dependent block of Nav and Cav channels at the atomic level. Our proposed experiments have three goals. 1. We will build upon strong preliminary data to reveal the high-resolution structure of the therapeutically important benzothiazepine diltiazem bound to its receptor site in the pore of CavAb, compare the chemistry of its binding to verapamil, determine the role of fenestrations in state-dependent block of CavAb, and explore the effects of mutations that substitute human residues in the AAD receptor site. 2. We will build on strong preliminary data to reveal the high- resolution structures of Class 1 AADs such as lidocaine and flecainide bound to NavAb, differentiate among the binding poses and receptor site conformations for Subclass 1A, 1B, and 1C AADs, determine the role of fenestrations in state- dependent block of NavAb, and explore the effects of mutations that humanize the NavAb drug receptor. 3. Based on a new homogeneous biochemical preparation, we will use cryo-electron microscopy and X-ray crystallography to determine the structure of a mammalian cardiac Nav1.5 channel at high resolution, define the structural basis for its unique physiological properties, and elucidate the structural basis for AAD block of Nav1.5 channels. Our results will be crucial for understanding and improving therapy of life-threatening cardiac arrhythmias by AADs.